Ischemia with or without reperfusion occurs in a variety of diseases including myocardial infarction, stroke, hemorrhagic shock and organ transplantation. We and others have shown that ischemia followed by reperfusion can result in a neutrophil (PMN)-induced reperfusion injury and that a portion of the injury can be ameliorated with monoclonal antibodies that recognize leukocyte adhesion molecules. These pre-clinical experiments led to a number of clinical trials using anti-adhesion therapy in an attempt to reduce injury following myocardial infarction, stroke and traumatic injury. Unfortunately, these trials did not demonstrate a protective effect in spite of the very strong pre-clinical data. In an attempt to understand these clinical studies, we examined ischemia times in the experimental setting of myocardial and cerebral ischemia-reperfusion that led to the clinical trials and found ischemia times to be generally less than 1.5 hours with the majority of times being between 30 and 60 min. It is possible that failure of the clinical trials can be explained by the extended ischemia times that occur in the clinical setting. Thus, in preliminary experiments, we increased the ischemia time up to 2 hours in hind limb ischemia and reperfusion in the mouse and found that there was indeed a CD18 adhesion molecule-dependent injury up to 60 minutes. However, when the ischemia time was increased to 90 minutes, the CD18-dependent portion of injury was completely eliminated. Still, treatment with the broad spectrum cysteine protease inhibitor z-VAD or by blocking complement activation protected from injury even for these extended ischemia times. Thus, we propose to examine mechanisms other than anti-adhesion therapy for the treatment of extended ischemia followed by reperfusion. In this application we propose the following specific aims: 1) To determine: a) whether leukocytes contribute to tissue injury when the ischemia time is sufficient to result in CD18-independent injury, and b) the length of ischemia that can be rescued by blocking caspase activation. 2) To determine the effect of Bcl-2 over-expression in various cell types (myeloid, lymphoid, muscle, endothelial) on the injury following extended ischemia and reperfusion. 3) To determine the role of the death receptors Fas and TNF, R1 in DNA strand-breaks, caspase activation and muscle injury following extended ischemia and reperfusion. 4) To determine the role of endothelial cell apoptosis following extended ischemia and subsequent reperfusion.